Improvements in recombinant DNA technology have produced dramatic changes to the nature of pharmaceutical and agricultural industries. One area where this technology has had great impact is in the development of diagnostics, in particular diagnostics to determine the suitability of candidates for IVF regimens. Traditionally, there has been no efficient means of predicting the outcome of an IVF programme, which is a costly procedure. Furthermore, recombinant DNA technology has led to advances in the development of diagnostics for assessing the hypertensive state of patients.
Corticosteroids, also referred to as glucocorticoids are steroid hormones, the most common form of which is cortisol. Modulation of glucocorticoid activity is important in regulating physiological processes in a wide range of tissues and organs. Glucocorticoids act within the gonads to directly suppress testosterone production (Monder et al., 1994). High levels of glucocorticoids may also result in excessive salt and water retention by the kidneys, producing high blood pressure.
Glucocorticoid action is mediated via binding of the molecule to a receptor, defined hereinafter as either a mineralocorticoid receptor (MR) or a glucocorticoid receptor (GR). Krozowski et al.(1983) and Beaumont and Fanestil (1983) showed that MR of adrenalectomised rats have an equal affinity for the mineralocorticoid aldosterone and glucocorticoids, for example corticosterone and cortisol. Confirmatory evidence has been found for human MR (Arriza et al., 1988). In patients suffering from the congenital syndrome of Apparent Mineralocorticoid Excess (AME; Ulick et al., 1979), cortisol levels are elevated and bind to and activate MRs normally occupied by aldosterone, the steroid that regulates salt and water balance in the body. Salt and water are retained in AME patients causing severe hypertension.
The enzyme 11 .beta.-hydroxysteroid dehydrogenase (11 .beta.HSD) converts glucocorticoids into metabolites that are unable to bind to MRs (Edwards et al., 1988; Funder et al., 1988), present in mineralocorticoid target tissues, for example kidney, pancreas, small intestine, colon, as well as the hippocampus, placenta and gonads. For example, in aldosterone target tissues 11 .beta.HSD inactivates glucocorticoid molecules, allowing the much lower circulating levels of aldosterone to maintain renal homeostasis. When the 11 .beta.HSD enzyme is inactivated, for example in AME patients (Ulick et al., 1979) or following administration of glycyrrhetinic acid, a component of licorice, severe hypertension results. Further, placental 11 .beta.HSD activity may protect the foetus from high circulating levels of glucocorticoid which may predispose to hypertension in later life (Edwards et al., 1993).
Although the 11 .beta.HSD enzyme has never been purified to homogeneity, biochemical characterisation of 11 .beta.HSD activity indicates the presence of at least two isoenzymes (11 .beta.HSD1 and 11 .beta.HSD2) with different cofactor requirements and substrate affinities. The 11 .beta.HSD1 enzyme is a low affinity enzyme that prefers NADP+ as a cofactor (Agarwal et al., 1989). The 11 .beta.HSD2 enzyme is a high affinity enzyme (Km for glucocorticoid=10 nM), requiring NAD+, not NADP+ as the preferred cofactor, belonging to a class of glucocorticoid dehydrogenase enzymes hereinafter referred to as"NAD+ dependent glucocorticoid dehydrogenase" enzymes. p Michael et al. (1993) show an inverse correlation between 11 .beta.HSD enzyme activity in human granulosa-lutein cells and the success of IVF, and suggest that activity of this enzyme might be related to the success of embryo attachment and implantation following IVF. The measurement of ovarian 11 .beta.HSD enzyme activity as a prognostic indicator for the outcome of assisted conception in all species, is the subject of UK Patent Application No 9305984. However, the disclosure of Michael et al. (1993), and corresponding UK Patent Application No 9305984 do not identify, or even suggest which isoenzyme in the ovary might be a predictive indicator of IVF embryo transfer, or a means of distinguishing isoenzymes of 11 .beta.HSD in the prediction of IVF embryo transfer outcomes. In fact, the enzyme assay procedure might detect all isoenzymes of 11 .beta.HSD activity in the cell, some of which may be hitherto uncharacterised.
Clearly, there are direct benefits to be derived from interventionary measures that modulate glucocorticoid levels in humans, for example using gene therapies. The gene encoding the NADP+ dependent glucocorticoid dehydrogenase enzyme 11 .beta.HSD1, has been cloned from rat cells (Agarwal et al., 1989). Analysis of the 11 .beta.HSD1 gene from AME patients (Nikknia et al., 1993) has failed to identify a potential correlation between activity of this particular genetic sequence and the syndrome. Furthermore, the isolation of the human 11 .beta.HSD1 gene has not contributed to the identification of other genes that do in fact play a role in the aetiology of diseases associated with glucocorticoid metabolism, for example AME. In particular, the molecular characterisation of a gene encoding the 11 .beta.HSD2 enzyme from human kidney cells, has not been a straightforward procedure. For example, the enzyme has not been purified to homogeneity and, until the present invention, there were no nucleotide sequence or amino acid sequence data for the 11 .beta.HSD2 enzyme from any primate species, including man.